This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Large regions of eukaryotic chromosomes are transcriptionally repressed due to DNA packaging in heterochromatin, a structure that is heritably propagated from one mitotic cycle to the next. Other chromosomal regions are transcriptionally active for brief periods during early development only to be shut-off permanently by repressive structures related to heterochromatin. Work in the Gartenberg lab has focused on mechanisms of heterochromatic gene repression in budding yeast. The hallmark of our approach over the last decade has been the use of site-specific recombination to physically uncouple silenced loci from other chromosomal sequences and activities. This has led to discoveries regarding the role of cis-acting sequences, DNA replication and nuclear localization in silencing. It did not escape our attention that liberating pieces of heterochromatin might facilitate their purification. Previously we developed a differential centrifugation approach to biochemically enrich DNA circles formed by site-specific recombination in vivo. We showed that the isolated material retained characteristics of silenced chromatin. We have since engineered a tandem affinity purification reagent to further purify the rings. In this collaboration with the John Yates lab we aim to identify the protein constituents of yeast heterochromatin by mass spectrometry.